The RNA interactome necessary and sufficient for Orbivirus genome packaging

As the current situation with the outbreak of SARS-CoV-2 demonstrates, virus infections represent an ever-present threat to the biosphere which ultimately has a human cost in terms of lives lost or economies compromised. Of all infectious organisms, viruses are the least well controlled as their replication is intimately tied-up with that of the host and the definition of components that are unique to the virus, as has been done for the development of antibiotics for bacteria, is often very challenging. Once viruses infect a host cell (human, animal or plant) replication is rapidly initiated and the virus makes copies of its proteins and genome and assembles them into progeny viruses in order to spread. An ideal target to inhibit this process would be one which is absolutely required and shared by all members of the same family. One such process is that of genome assembly. The process of virus synthesis is only possible if the viral genome is incorporated into nascent progeny in the correct form and it is particularly difficult for viruses to incorporate the genome if it is in multiple pieces, which is the case for many viruses of concern (e.g. influenza virus). Understanding how multiple pieces of genomes, each of a different sequence, are incorporated in the correct order and form is essential if progress is to made on how viruses multiply and spread. The lack of this information has created a bottleneck in the research and understanding of many viruses over the years. This project focuses on an animal virus, Bluetongue virus (BTV), a notable pathogen of sheep and cattle. BTV and related viruses are highly pathogenic in animals and humans with BTV killing livestock with devastating economic consequences. The viruses are characterized by their 10 or 11 genomic pieces of RNA known as genomic segments, each representing a single gene and each generating specific viral proteins. This project will investigate the most vital process of how new RNA pieces are incorporated into the virus particle during the replication cycle, which will offer new opportunities for designed intervention and the development of attenuated vaccines for the control of disease. Further, the understanding of this fundamental process also underpins much RNA biology and impinges on a wide range of applications, from understanding RNA disease to the development of advanced therapies in which novel RNAs are incorporated into viruses for the purposes of gene delivery and gene therapy.

The group is exceptionally positioned at the forefront of this field of research as a result of previous BBSRC funded projects in which we developed unique tools, techniques and reagents. With a series of novel assay systems we have already defined some of the essential steps of how BTV may recruit their segments and assemble into a viable virus, competent for spreading. The precise nature of selection and packaging, that is acquiring a single copy of each of ten segments rather than multiple copies of one, is a complex and highly regulated process as the virus must differentiate between cellular and viral genes.
Long term improvements in animal welfare underpinned by basic research into the pathogen concerned are important. BTV is highly pathogenic in certain livestock and has recently emerged in the UK and Europe. Understanding these vital basic processes in the life cycle of the viral genome will make it possible to develop novel, safer designer vaccines for Bluetongue disease and may be other viral diseases that affect livestock. The data will also allow for the development of novel interventions to improve future disease management.

The genome defines the organism and how genomes are configured and packaged is therefore a fundamental question in biology. It is a particular challenge for viruses with segmented genomes, such as the 10-segment dsRNA Bluetongue virus (BTV), as any mechanism much be capable of "counting" one copy of each segment into progeny virus particles. In addition, the packaged genome cannot be too constrained as it must be able to be released quickly upon infection of a new cell. Thus, a balance must be struck between a mechanism that is accurate, rigidly encases and protects the RNA and being sufficiently flexible to allow it to act as a template. This complexity has kept the detail of the mechanism undetermined to the present time yet its decoding could provide many new opportunities for therapeutic intervention. In this project we will identify the RNA-RNA and RNA-protein interactions required for packaging in BTV. The project has direct relevance to pathogen control as BTV is a notable pathogen for livestock, being responsible for significant economic losses worldwide but also more generally as BTV acts as a model for other dsRNA viruses of clinical significance. We will use our recently published bespoke assay systems to interrogate the sequential steps involved in RNA complex formation. We will map the RNA-RNA points of contact in vitro and in vivo and determine the extent to which they obey mathematical predications. We will determine which proteins act in-trans on such RNA complexes and how this achieves the selective recruitment of the RNA genome into the assembling virus. During the project we will use many novel assays including high-throughput CLIP-Seq, SHAPE/SPLASH and established reverse genetics systems to determine how RNAs interact and the changes that occur when viral proteins bind to ssRNAs and how they alter RNA fate. Our goal is to provide a thorough understanding of genome packaging at the molecular and structural levels for BTV and for related viruses.